EP3846848A1 - Vaccins atténués vivants à base de plasmide d'adn pour virus à arn simple brin sens positif - Google Patents

Vaccins atténués vivants à base de plasmide d'adn pour virus à arn simple brin sens positif

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Publication number
EP3846848A1
EP3846848A1 EP19857888.2A EP19857888A EP3846848A1 EP 3846848 A1 EP3846848 A1 EP 3846848A1 EP 19857888 A EP19857888 A EP 19857888A EP 3846848 A1 EP3846848 A1 EP 3846848A1
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EP
European Patent Office
Prior art keywords
virus
attenuated
zikv
live
dna
Prior art date
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Pending
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EP19857888.2A
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German (de)
English (en)
Other versions
EP3846848A4 (fr
Inventor
Jing Zou
Xuping XIE
Pei-Yong Shi
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University of Texas System
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University of Texas System
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Publication of EP3846848A1 publication Critical patent/EP3846848A1/fr
Publication of EP3846848A4 publication Critical patent/EP3846848A4/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24171—Demonstrated in vivo effect
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention generally relates to the development of a DNA-launched live- attenuated vaccine (LAV) for plus-sense single stranded RNA viruses and
  • LAV live- attenuated vaccine
  • compositions comprising the vaccine.
  • the vaccines may be used in humans and animals for treating or providing immunoprotection against plus-sense single stranded RNA viruses such as ZIKA virus (ZIKV), yellow fever virus and Japanese encephalitis.
  • the genus Flavivirus of the family Flaviviridae comprises over 70 viruses, many of which are arthropod-borne and significant viral pathogens, including dengue (DENV), yellow fever (YFV), Japanese encephalitis (JEV), West Nile (WNV), Zika (ZIKV), St. Louis encephalitis (SLEV), and tick-borne encephalitis (TBEV) viruses.
  • DENV dengue
  • YFV yellow fever
  • JEV Japanese encephalitis
  • WNV West Nile
  • ZIKV Zika
  • SLEV St. Louis encephalitis
  • TBEV tick-borne encephalitis
  • the four serotypes of DENV alone affect over 3 billion people living in the tropics and subtropics, leading to 390 million human infections each year [48].
  • YFV poses risks to 900 million people living in 44 endemic countries of Latin America and Africa and kills around 60,000 people annually [49] JEV is the most common cause
  • ZIKV congenital Zika syndromes
  • Flaviviruses have a positive-sense, single-stranded RNA genome of about 11 ,000 nucleotides.
  • the viral genome consists of a 5' untranslated region (UTR), a single open-reading frame, and a 3' UTR.
  • the long open-reading frame encodes three structural (capsid [C], precursor membrane [prM], and envelope [E]) and seven non-structural (NS1 , NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. Together with the genomic RNA, the structural proteins form viral particles. The nonstructural proteins participate in viral replication, virion assembly, and evasion of host immune response [54] Reverse genetic systems have been well developed for different flaviviruses to study viral replication, pathogenesis, vaccine, and antiviral development [55-61]
  • Vaccines have saved millions of lives through controlling and preventing infectious diseases. Compared with the inactivated and subunit vaccines, live- attenuated vaccines (LAVs) have the advantage of rapid immune response, potentially single dose, and durable protection.
  • Successful LAVs for viral pathogens include smallpox, polio, measles, mumps, rubella, rotavirus, chickenpox, yellow fever, Japanese encephalitis 14-14-2, and influenza nasal spray vaccines.
  • the LAVs are usually manufactured in cells, eggs, or animals and transported to clinics through a“cold chain.” The LAV production in cells or eggs requires dedicated manufacture facility and has the risk of adventitious agent contamination.
  • RNA platform has recently emerged as a promising means to launch non-replicative RNA or self- amplifying viral RNA (i.e., LAV or replicon) [63]. Once the LAV or replicon RNA is delivered into cells, it translates and replicates, leading to efficient expression of antigens.
  • the Flavivirus genome is about 11 ,000 nucleotides in length and contains a 5' untranslated region (UTR), a long open-reading frame, and a 3' UTR.
  • the single open-reading frame encodes three structural (capsid [C], precursor membrane [prM] and envelope [E]) and seven non-structural (NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins.
  • the structural proteins together with the genomic RNA, form viral particles.
  • the nonstructural proteins participate in viral replication, virion assembly, and evasion of the host innate immune response [1]
  • the flaviviral 3' UTR ranges in length from about 400 to 600 nucleotides, and is highly structured with regions conserved between species.
  • ZIKV was first identified from a sentinel rhesus macaque in the Zika Forest of Kenya in 1947 [2] Before 2007, ZIKV had silently circulated between primates and mosquitoes in the forests in Africa and Southeast Asia without causing detectable outbreaks or severe human diseases.
  • Symptomatic ZIKV infection produces mild manifestations, such as fever, headaches, lethargy, conjunctivitis, rash, arthralgia, and myalgia [3]
  • ZIKV emerged explosively to cause a series of epidemics in Africa, Micronesia, the South Pacific, and the Americas, leading to more than 700,000 documented autochthonous human infections [4, 5]
  • ZIKV caused the newly described devastating congenital Zika syndromes (CZS), including microcephaly, craniofacial disproportion, spasticity, ocular abnormalities, and miscarriage [6]
  • CZS was found in 6-11 % of the fetuses from ZIKV-infected pregnant women [7]
  • Zika infection can cause Guillain-Barre syndrome (GBS; an autoimmune disease that leads to muscle weakness and paralysis) at an incidence of 1 in 4,000-to-5,000 infected adults [8]
  • GBS Guillain-
  • Subunit vaccines express the viral prM-E proteins from DNA, mRNA, or viral vectors (including measles virus, vesicular stomatitis virus, adenovirus, and modified vaccinia virus) [14-19]
  • viral vectors including measles virus, vesicular stomatitis virus, adenovirus, and modified vaccinia virus.
  • LAV Live-attenuated vaccine
  • DNA vaccines are chemically stable and do not require a cold chain.
  • traditional DNA vaccines expressing viral antigens usually require multiple doses, and the immune responses observed in animal models have generally not been reproduced in humans [26]
  • LAVs usually have the advantages of single dose, quick immunity, and durable protection.
  • the manufacture and transport of LAVs require cell culture (or eggs) and a cold chain. The cold chain alone can account for 80% of the vaccine cost in warm climates where emerging viruses are typically endemic [27]
  • a DNA-launched LAV has the potential to combine the strengths and to eliminate the weaknesses of both vaccine platforms. These improvements are of practical importance and could transform future vaccine development.
  • Vaccines especially live-attenuated vaccines (LAV) are the most effective way to control and prevent infectious diseases.
  • LAVs have the advantages of single dose, rapid immune onset, and long-lasting protection.
  • DNA vaccine has the advantages of chemical stability, ease of production, and no“cold chain” requirement during storage and transportation. Combining the strengths of LAV and DNA vaccine could transform the practice of conventional LAV by eliminating vaccine production on cell culture (or eggs) or“cold chain” requirement.
  • pLAV plasmid DNA-launched LAV
  • the present invention addresses the need for providing methods for producing live attenuated vaccines of improved stability and immunogenicity against positive-strand RNA viruses with global public health impact such as flaviviruses.
  • the invention relates to an immunogenic composition
  • an immunogenic composition comprising a cDNA copy of the genome of a live-attenuated plus-sense single-stranded RNA virus wherein such viral genome optionally comprises at least one mutation which attenuates or reduces the virulency of said single-stranded RNA virus in a
  • susceptible host e.g , wherein said mutations include substitution, deletion and addition mutations which elicit the production of neutralizing antibodies in a susceptible host and/or elicit a protective T cell immune response in a susceptible host.
  • the invention relates to an immunogenic composition as above-described wherein the live-attenuated plus-sense single-stranded RNA virus is a live- attenuated flavivirus, e.g., wherein the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated ZIKA virus (ZIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), or tick-borne encephalitis virus (TBEV) or comprises a live-attenuated plus-sense single-stranded RNA virus is a live-attenuated ZIKA virus (ZIKV) and/or wherein the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated yellow fever virus (YFV) and/or wherein the live-attenuated plus-sense single-stranded RNA virus is a live- attenu
  • the invention further relates to the immunogenic composition of any one of the foregoing, wherein the genome of the live-attenuated plus-sense single-stranded RNA virus comprises a deletion in the 3’ untranslated region (3’UTR) of the genome which attenuates or reduces the virulency of said single-stranded RNA virus in a susceptible host, e.g., wherein the genome of the live-attenuated plus-sense single- stranded RNA virus comprises a deletion in the 3’ untranslated region (3’UTR) of the genome which attenuates or reduces the virulency of said single-stranded RNA virus in a susceptible host and/or comprising a 3’UTR deletion ranges in size from about 5 to 40 nucleotides and/or comprising a 3’UTR deletion which comprises a 10- nucleotide deletion, a 20-nucleotide deletion, or a 30-nucleotide deletion ⁇ 1 , 2, 3, 4,
  • the invention further relates to the immunogenic composition of any one of the foregoing, wherein the genome of the live-attenuated plus-sense single-stranded RNA virus in the plasmid comprises the genome of an attenuated JEV strain, e.g., JE14-14-2 having the sequence in FIG. 18 (SEQ ID NO: 1) or comprises the genome of an attenuated YFV strain, e.g., YF17D and optionally expresses the LAV under the control of a eukaryotic or viral promotor.
  • the genome of the live-attenuated plus-sense single-stranded RNA virus in the plasmid comprises the genome of an attenuated JEV strain, e.g., JE14-14-2 having the sequence in FIG. 18 (SEQ ID NO: 1) or comprises the genome of an attenuated YFV strain, e.g., YF17D and optionally expresses the LAV under the control of a eukaryotic
  • the invention further relates to the immunogenic composition of any one of the foregoing, which comprises at least one pharmaceutically acceptable carrier or excipient and/or adjuvant, e.g., one suitable for parenteral or enteral administration and/or via injection such as intramuscular, intravenous, subcutaneous and/or optionally further including a subsequent electroporation.
  • at least one pharmaceutically acceptable carrier or excipient and/or adjuvant e.g., one suitable for parenteral or enteral administration and/or via injection such as intramuscular, intravenous, subcutaneous and/or optionally further including a subsequent electroporation.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing which (i) induces a CD8+ T cell response, an antibody response, and/or a cellular immune response against the plus-sense single stranded RNA virus; (ii) produces a neutralizing antibody titer equivalent to that of wildtype plus-sense single stranded RNA virus infection; and/or (iv) prevents viremia in a susceptible subject after subsequent challenge with a wildtype plus-sense single stranded RNA virus strain.
  • an immunogenic composition according to any one of the foregoing which (i) induces a CD8+ T cell response, an antibody response, and/or a cellular immune response against the plus-sense single stranded RNA virus; (ii) produces a neutralizing antibody titer equivalent to that of wildtype plus-sense single stranded
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing, which (i) induces a CD8+ T cell response, an antibody response, and/or a cellular immune response against ZIKV; (ii) produces a neutralizing antibody titer equivalent to that of wildtype ZIKV infection; (iii) is used to prevent congenital ZIKV syndrome and/or microcephaly; and/or (iv) prevents viremia in said subject after subsequent challenge with a wildtype ZIKV strain.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing, which (i) induces a CD8+ T cell response, an antibody response, and/or a cellular immune response against JEV; (ii) produces a neutralizing antibody titer equivalent to that of wildtype JEV infection; (iii) is used to prevent JEV mediated encephalitis or weight loss; and/or (iv) prevents viremia in said subject after subsequent challenge with a wildtype JEV strain or JE14-14-2.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing which (i) induces a CD8+ T cell response, an antibody response, and/or a cellular immune response against YFV; (ii) produces a neutralizing antibody titer equivalent to that of wildtype YFV infection; (iii) is used to prevent YFV mediated fever or weight loss; and/or (iv) prevents viremia in said subject after subsequent challenge with a wildtype YFV strain or YFV17D.
  • an immunogenic composition according to any one of the foregoing which (i) induces a CD8+ T cell response, an antibody response, and/or a cellular immune response against YFV; (ii) produces a neutralizing antibody titer equivalent to that of wildtype YFV infection; (iii) is used to prevent YFV
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing wherein the susceptible subject is a human and/or a pregnant female.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing, wherein the susceptible subject has never been exposed to the virus.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing, wherein the susceptible subject has been previously exposed to the virus.
  • the invention further relates to a method of eliciting an immune response in a susceptible subject by administering a prophylactically or therapeutically effective amount of an immunogenic composition according to any one of the foregoing, wherein the administered immunogenic composition comprises cDNA copies of the genome of different live-attenuated plus-sense single-stranded RNA viruses wherein each of said different viral genomes optionally comprises at least one mutation which attenuates or reduces the virulency of said single-stranded RNA virus in a
  • RNA viruses in the administered immunogenic composition are derived from different flaviviruses and/or different strains of a specific flavivirus, e.g., ZIKV, YFV, or JEV.
  • the invention further relates to methods of providing prolonged immunity against a flavivirus comprising administering a single or multiple doses of a plasmid comprising a cDNA encoding live attenuated strain of said flavivirus.
  • the invention further relates to methods of providing prolonged immunity against YFV, JEV or ZIKV comprising administering a single or multiple doses of a plasmid comprising a cDNA encoding a live attenuated strain of ZIKV, YFV, or JEV, e.g., wherein the attenuated virus is YFV and the plasmid comprises a cDNA encoding YF17D (accession number JN628279.1) which optionally comprises at least one mutation and/or the viral cDNA in the plasmid comprises JE14-14-2, optionally which comprises at least one mutation optionally a K136E or K166Q mutation.
  • the invention further relates to methods of providing prolonged immunity against YFV, JEV or ZIKV comprising administering a single or multiple doses of a plasmid comprising a cDNA encoding a live attenuated strain of a flavivirus, e.g., ZIKV, YFV, or JEV, wherein the virus cDNA is expressed under the control of a eukaryotic or viral promoter, e.g., the SV40 promoter and/or comprises a hepatitis delta virus ribozyme (FIDVr) and a polyadenylation signal engineered at the 3’ end of the viral cDNA and/or the plasmid comprising the virus cDNA is one that provides for low copy number and optionally copy number control, e.g., pCC1.
  • a eukaryotic or viral promoter e.g., the SV40 promoter and/or comprises a hepatitis delta virus ribozyme (FI
  • the invention further relates to methods of providing prolonged immunity against YFV, JEV or ZIKV comprising administering a single or multiple doses of a plasmid comprising a cDNA encoding a live attenuated strain of ZIKV, YFV, or JEV, wherein the plasmid comprising the cDNA encoding the attenuated viral DNA flavivirus is administered by injection, e.g., intramuscularly and optionally further including an electroporation step after injection.
  • the invention further relates to methods of providing prolonged immunity against YFV, JEV or ZIKV comprising administering a single or multiple doses of a plasmid comprising a cDNA encoding a live attenuated strain of ZIKV, YFV, or JEV, wherein the virus immunizing dose is as low as 300 ng or 500 ng of pLAV.
  • the at least one mutation which attenuates or reduces the virulency of said single-stranded RNA virus in a susceptible host includes one or more substitution, deletion and addition mutations.
  • the immunogenic composition elicits the production of neutralizing antibodies in a susceptible host.
  • the immunogenic composition elicits a protective T cell immune response in a susceptible host.
  • the immunogenic composition elicits the production of neutralizing antibodies and a protective T cell immune response in a susceptible host.
  • the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated flavivirus.
  • the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated ZIKA virus (ZIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), or tick-borne encephalitis virus (TBEV).
  • ZIKV live-attenuated ZIKA virus
  • DEV dengue virus
  • YFV yellow fever virus
  • WNV West Nile virus
  • JEV Japanese encephalitis virus
  • TBEV tick-borne encephalitis virus
  • the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated yellow fever virus, e.g., derived from clinical strain YF17D or a variant thereof.
  • the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated Japanese encephalitis virus, e.g., clinical strain JE14- 14-2 or a variant thereof.
  • the live-attenuated plus-sense single-stranded RNA virus is a live-attenuated ZIKA virus (ZIKV).
  • the genome of the live-attenuated plus-sense single- stranded RNA virus comprises a deletion in the 3’ untranslated region (3’UTR) of the genome which attenuates or reduces the virulency of said single-stranded RNA virus in a susceptible host
  • the 3'UTR deletion ranges in size from about 5 to 40 nucleotides.
  • the 3'UTR deletion comprises a 10-nucleotide deletion, a 20-nucleotide deletion, or a 30-nucleotide deletion ⁇ 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.
  • the 3’UTR deletion is a 20-nucleotide deletion.
  • the 3’UTR deletion is a 20-nucleotide deletion deletion of the nucleotides at positions 10640 to 10659 of the full-length genomic sequence.
  • the cDNA copy is comprised in a DNA plasmid.
  • the DNA plasmid is an expression vector comprising a eukaryotic or viral promotor.
  • the immunogenic composition comprises at least one pharmaceutically acceptable carrier or excipient and/or adjuvant.
  • the immunogenic composition is suitable for parenteral or enteral administration.
  • the administered immunogenic composition comprises cDNA copies of the genome of different live-attenuated plus-sense single-stranded RNA viruses wherein each of said different viral genomes optionally comprises at least one mutation which attenuates or reduces the virulency of said single-stranded RNA virus in a susceptible host.
  • the genomes of the different live-attenuated plus-sense single-stranded RNA viruses in the administered immunogenic composition are derived from different flaviviruses and/or different strains of a specific flavivirus, e.g., ZIKV, Japanese encephalitis virus and/or yellow fever virus.
  • FIG. 1A-E contains a characterization of pZIKV-3’UTR-A20 in cell culture.
  • A Diagram of plasmid pZIKV-3’UTR-A20.
  • the plasmid pCC1TM vector was used to engineer a gene cassette containing a promoter from simian virus 40 (SV40), ZIKV- 3’UTR-A20 cDNA, hepatitis delta virus ribozyme (HDVr) sequence, and SV40 polyadenylation (pA) signal element. Junction sequences are depicted between the SV40 promoter and the 5’UTR of viral genome.
  • the 20-nucleotide deletion at the 3’UTR of ZIKV genome is indicated by a dotted line and nucleotide positions
  • Vero cells were transfected with pZIKV-WT, pZIKV-3’UTR-A20 (D20), or pZIKV-AGDD (AGDD). At the indicated time post-transfection (p.t), the cells were stained with 4G2 antibody to detect viral E protein expression (green). Nuclei were
  • D Virus production post-transfection. Supernatants from the transfected Vero cells were quantified for infectious ZIKV using a focus-forming assay. The dotted line indicates the limit of detection (L.O.D.) of 10 FFU/ml. Multiple t-test was performed to analyze the statistical significances.
  • E Focus-forming morphologies of WT ZIKV and ZIKV- 3’UTR-A20 virus. No infectious virus was detected from the pZIKV-AGDD- transfected cells.
  • FIG. 2A-K shows that immunization of pZIKV-3’UTR-A20 protects the A129 mouse from ZIKV challenge.
  • A Experimental design. Various doses of pZIKV- 3’UTR-A20 (1 , 0.1 , 0.01 pg), pZIKV-AGDD (10 pg), or DPBS (sham) were
  • mice were monitored for weight loss over 14 days. Since our IACUC protocol only allows four blood draws per mouse over 28 days post-transfection (or infection), blood draws were staggered for different mouse sub-cohorts to cover the sampling period of days 5-10 post-immunization. Mice were bled at indicated time for measuring neutralizing antibody titers (NT50) using a mCherry-ZIKV neutralization assay. On day 29 post immunization, the mice were challenged with 10 6 PFU of ZIKV strain PRVABC59 via the subcutaneous route (indicated by a red arrow). At indicated time, the mice were bled for measuring viremia using a focus-forming assay.
  • NT50 neutralizing antibody titers
  • C-F Viremia for the mouse groups immunized with 1 pg pZIKV- 3’UTR-A20 (c), 0.1 pg pZIKV-3’UTR-A20 (D), 0.01 pg pZIKV-3’UTR-A20 (E), or 10 pg pZIKV-AGDD (F).
  • G-J Neutralizing antibody titers (NT50) from the mouse groups immunized with 1 pg pZIKV-3’UTR-A20 (G), 0.1 pg pZIKV-3’UTR-A20 (H), 0.01 pg pZI KV-3’ UTR-A20 (I), or 10 pg pZIKV-AGDD (J). Group sizes (n number) are indicated. Individual mice are indicated by different colors and symbols. Paired t-test was performed to indicate no significant difference (n.s.) between the prechallenge (day 29) and post-challenge (day 43) neutralizing antibody titers in (G).
  • K Summary of viremia-positive and neutralizing antibody-positive ratios for all mouse groups. Limits of detections (L.O.D., dotted lines) of focus-forming assay and neutralization assay (NT50) were 100 FFU/ml and 100 dilution, respectively.
  • FIG. 3A-I shows the safety of pZIKV-3’UTR-A20 in male mice.
  • mice Six-week-old male A129 mice were immunized with pZIKV- 3’UTR-A20 (1 pg), pZIKV-WT (1 pg), or DPBS (sham) via intramuscular (IM) injection and electroporation (EP) using TriGridTM. On day 29 post-immunization, mice were sacrificed for analysis. Neutralizing antibody titers were measured on day 29 post-immunization using an mCherry ZIKV neutralization assay (B). Viral loads in mouse brain (C), spleen (D), and testis (E) were determined by a focus-forming assay.
  • the L.O.D.s for organ viral load and NTso were 100 FFU/g of tissue and 100 dilution, respectively.
  • F Testis weight.
  • G Representative images of testes from each group. The epididymis was harvested for counting total sperm (H) and motile sperm (I). Individual mice are indicated by different colors and symbols. The means and standard deviations are shown. A one-way analysis of variance (ANOVA) test was performed to determine the statistically significant differences among groups.
  • FIG. 4A-J shows immunization with pZ!KV-3’UTR-A20 protects male mice from ZIKV-induced damages to testis.
  • A Experimental design. The bottom panel shows three mouse groups (I, II, and III) with different immunizing agents and challenge conditions. Six-week-old male A129 mice were vaccinated with pZIKV- 3’UTR-A20 (1 pg) or DPBS (sham) using TriGridTM. On day 29 post-immunization, the mice were challenged with 10 6 PFU of epidemic ZIKV strain PRVABC59 or DPBS controls.
  • FIG. 5A-I illustrates prevention of vertical transmission from pregnant mice.
  • A Experimental design. The right panel shows three mouse groups (IV, V, and VI) with different immunizing agents and challenge conditions. Six-week-old female A129 mice were immunized with pZIKV-3’UTR-A20 (1 pg) or DPBS (sham) using TriGridTM. At E10.5, mice were challenged with 10 6 PFU of ZIKV strain PRVABC59 or DPBS controls via the subcutaneous route. At E18.5, the mice were sacrificed for measuring viral loads in maternal and fetal organs.
  • B Maternal NT50 values on day 29 post-immunization and at E18.5.
  • paired t-test was performed to indicate no significant difference (n.s.) between the pre-challenge (day 29) and post-challenge (E18.5) neutralizing antibody titers.
  • C Viremia on day two post-challenge.
  • D Maternal brain viral loads.
  • E Maternal spleen viral loads.
  • F Placenta viral loads.
  • G Fetal head viral loads.
  • H Fetal weights.
  • I Neutralizing antibodies in fetal blood. Individual dams are indicated by different colors and symbols. Fetuses and their parental mice are matched with the same colors and symbols.
  • the L.O.D.s for viremia, organ virus load, and neutralizing antibody titer are 100 PFU/ml, 100 PFU/g, and 100 dilutions, respectively.
  • a one-way ANOVA test was performed to determine statistically significant differences among groups.
  • FIG. 6A-E shows T cell responses in A129 mice after pZIKV-3’UTR-A20 immunization.
  • Six-week-old A129 mice were immunized with pZIKV-3’UTR-A20 (0.5 pg) or DPBS (sham) using TriGridTM.
  • TriGridTM TriGridTM
  • splenocytes were harvested for T cell analysis.
  • A Total numbers of CD4 + T cell subsets per spleen. Splenocytes were cultured ex vivo with ZIKV for 24 h and stained for IFN-y, TNF-a, and CD4 T cell markers.
  • B Total numbers of CD8 + T cell subsets.
  • Splenocytes were cultured ex vivo with ZIKV for 24 h (right panel) or with an E peptide for 5 h (left panel) and stained for IFN-g, TNF-a, and CD8 T cell markers.
  • Cytokines IL-2 (C), IFN-g (D), and TNF-a (E) in cell culture media were measured after splenocytes were stimulated by ZIKV for 2 days.
  • An unpaired nonparametric Mann-Whitney test was performed to analyze statistical significance.
  • FIG. 7A-B shows a comparison of SV40 and CMV promoters in pZIKV-WT to launch WT ZIKV replication in cell culture.
  • A Diagram of pZIKV-WT plasmid.
  • Plasmid pCC1TM vector was used to engineer a gene cassette containing a promoter from simian virus 40 (SV40) or from cytomegalovirus (CMV), WT ZIKV cDNA, hepatitis delta virus ribozyme (FIDVr) sequence, and SV40 polyadenylation (pA) signal element.
  • SV40 simian virus 40
  • CMV cytomegalovirus
  • FIDVr hepatitis delta virus ribozyme
  • pA SV40 polyadenylation
  • Plasmids pZIKV-WT containing SV40 promoter or CMV promoter (4 pg) were transfected into Vero (Top panel) or 293T (bottom panel) cells. Culture supernatants were collected daily and viral titers were determined by plaque assay. It should be noted that ZIKV
  • FSS13025 strain does not replicate efficiently in 293T cells.
  • the limited of detection (L.O.D.) of plaque assay is 10 PFU/ml, as indicated by the dotted line. Multiple t-test was performed to analyze the statistical significances.
  • FIG. 8A-F shows the minimal dose of pZIKV-3’UTR-A20 required for seroconversion and protection.
  • A Experimental design. Six-week-old A129 mice were immunized with 0.5 or 0.3 pg of pZIKV-3’UTR-A20 using TriGridTM. At the indicated time points, the mice were bled for measuring viremia and neutralizing antibody titers.
  • B Viremia from the 0.5 pg pZIKV-3’UTR-A20 group.
  • C Viremia from the 0.3 pg pZIKV-3’UTR-A20 group.
  • FIG. 9A-E contains a characterization of pZIKV-WT in the A129 mice.
  • A Experimental design. Six-week-old A129 mice were immunized with pZIKV-WT (1 pg) or DPBS (sham) using TriGridTM. Following immunization, the mice were monitored for weight loss over 14 days (B). At the indicated time points, the mice were bled for measuring viremia (C) and neutralizing antibody titers (D). Individual mice are indicated by different colors and symbols
  • E Comparison of the mean viremia between the pZIKV-WT- and pZIKV-3’UTR-A20 immunized mice.
  • the mean viremia curve for the pZIKV-WT-immunized mice was derived from (C) of this figure.
  • the mean viremia curve for the pZIKV-3’UTR-A20-immunized mice was derived from Fig. 2C.
  • FIG. 10A-F illustrates Efficiency of DNA delivery into the A129 mice by intramuscular (IM) needle injection without electroporation.
  • A Experimental design. Six-week-old A129 mice were immunized with pZIKV-3’UTR-A20 (1 pg), pZIKV-WT (1 pg), or DPBS (sham) by IM. Following immunization, the mice were monitored for weight loss over 14 days. The mice were bled on day 6-11 for measuring viremia and on day 29 for determining neutralizing antibody titers.
  • B Viremia in the pZIKV- WT-immunized mice.
  • D Neutralizing antibody titers from the pZIKV-WT-immunized mice on day 29 postimmunization.
  • E Neutralizing antibody titers from the pZIKV-3’UTR-A20- immunized mice on day 29 post-immunization. Individual mice are indicated by different colors and symbols.
  • FIG. 11A-C shows T cell response on day 29 post-immunization.
  • Splenocytes were cultured ex vivo with infectious ZIKV for 24 h or a ZIKV E peptide for 5 h, and stained for IFN-g, TNF-a, and T cell markers. The cells were then gated on CD4 + or CD8 + T cell subsets. Representative flow cytometry images are shown.
  • A CD4 + T cell subsets after ZIKV stimulation.
  • B CD8 + T cell subsets after the E peptide stimulation.
  • C CD8 + T cell subsets after ZIKV stimulation.
  • FIG. 12 contains a comparison of the relative sensitivity of plaque and RT- PCR assays. Different amounts of WT ZIKV were measured by plaque and RT-PCR assays. The plot shows the relative correlation and sensitivity of the two assays. Coefficient of determination (R 2 ) was determined using linear regression analysis. The limits of detection of plaque and RT-PCR assays are 10 PFU/ml and 500 RNA copy/ml, respectively.
  • FIG. 13A-D contains experimental results characterizing pYF17D and pJE14- 14-2 in cell culture.
  • A Plasmid scheme of pYF17D and pJE14-14-2.
  • Vector plasmid pCCITM was engineered with an SV40 promoter, YF17D or JE14-14-2 cDNA, hepatitis delta virus ribozyme (FIDVr), and SV40 polyadenylation (pA) signal sequence.
  • B-C LAV production in cell culture. Vero and BHK-21 cells were transfected with pYF17D and pJE14-14-2 DNA (4 pg), respectively. Culture supernatants were quantified for LAVs by plaque assay. Dotted lines indicate the limit of detection (L.O.D.).
  • D Plaque morphologies of YF17D and JE14-14-2 LAVs.
  • FIG. 14A-G contains experimental results characterizing JE14-14-2 and YF17D LAVs in A129 mice.
  • Nine-week-old A129 mice were subcutaneously infected with 10 4 PFU of JE14-14-2 or 105 PFU of YF17D LAV.
  • the infected mice were monitored for weight loss (A, D), mortality (B, F), and viremia (C, G).
  • Viremia was quantified by plaque assay on BHK-21 cells.
  • FIG. 15A-E contains experimental results relating to the vaccination and efficacy of pYF17D in A129 mice.
  • A Experimental design. Nine-week-old A129 mice were intramuscularly vaccinated with 1 , 0.3, or 0.1 pg of pYF17D using DNA delivery device TriGridTM. YF17D LAV and PBS were included as controls.
  • B Weight changes of vaccinated mice.
  • C Neutralizing antibody titers at days 14 and 21 post-vaccination. Neutralizing titers (NT50) were determined by sera dilutions that reduced 50% of the signal from nano luciferase YFV-17D reporter virus.
  • D Viremia protection after challenge.
  • mice On day 21 post-vaccination (p.v ), the mice were challenged with 107 PFU of YF17D virus. Peak viremia on day 2 post-challenge was quantified by plaque assay on BHK-21 cells.
  • E Post-challenge neutralizing antibody titers. On day 21 post-challenge (p.c.), mice were bled and measured for neutralizing titers using a nano luciferase YF17D reporter virus.
  • FIG. 16A-E contains experimental results relating to the vaccination and efficacy of pJE14-14-2 in A129 mice.
  • A Experimental design. Nine-week-old A129 mice were intramuscularly vaccinated with 1 , 0.3, or 0.1 pg of pJE14-14-2 using TriGridTM. JE14-14-2 LAV and PBS were included as controls.
  • B Weight changes of the vaccinated mice.
  • C Neutralizing antibody titers at days 14 and 21 postvaccination. NT50 values were determined using a nano luciferase JE14-14-2 reporter virus.
  • D Viremia protection after challenge. On day 21 post-vaccination, the mice were challenged with 10 4 PFU of JE14-14-2 virus.
  • FIG. 17A-D contains experimental results characterizing the stability of pYF17D and pJE14-14-2 in.
  • A Passaging scheme of E. coli containing pYF17D or pJE14-14-2.
  • coli colony containing pYF17D or pJE14-14-2 was inoculated into a 250-ml flask containing 50 ml LB medium with 12.5 pg/ml chloramphenicol, cultured at a 37°C for 12 h, and continuously cultured from P1 to P5 with 1000-fold dilution.
  • the E. coli culture from each passage was 10-fold diluted into fresh LB medium containing 12.5 pg/ml chloramphenicol and 1 x CopyControl Induction Solution. After 5 h incubation at 37°C, the culture was harvested for plasmid extraction. The resulting DNA plasmid was subjected to functional analysis.
  • FIG. 18 contains the genome sequence of JE14-14-2 used in Example 2.
  • FIG 19A-B contains the construction schemes used to synthesize pYF17D (a) and pJE14-14-2 (b).
  • Vector plasmid pCC1 with SV40 polyA sequence was derived from digesting pZIKV-3’UTR-A20 [75] with Hpal and Clal enzymes.
  • FIG 20A-B contains experimental results characterizing pJE14-14-2 on Vero cells.
  • A Production of JE14-14-2 LAV after transfecting Vero cells with 4 pg of pJE14-14-2 DNA. Culture supernatants were quantified for JE14-14-2 LAV by plaque assay on BHK-21 cells. The dotted line indicates the limit of detection (L.O.D.).
  • B Plaque morphology of JE14-14-2 virus recovered from Vero cells.
  • FIG 21A-E contains experimental results relating to heterogeneous JE14-14- 2 variants derived from Vero cells.
  • A Passage scheme of JE14-14-2 LAV on Vero cells. JE14-14-2 LAV was produced from pJE14-14-2-transfected Vero cells (named as P0). The P0 virus was continuously passaged on Vero cells for 5 rounds. The P5 virus was subjected to full-genome sequencing. Two independent passaging and sequencing were performed.
  • P0 pJE14-14-2-transfected Vero cells
  • the P5 virus was subjected to full-genome sequencing. Two independent passaging and sequencing were performed.
  • B Plaque morphologies of P0 to P5 JE14-14-2 on BHK-21 cells.
  • C Sequence chromatograms of the mutated E regions. The codons for K136E and K166Q are underlined.
  • FIG 22A-C contains experimental results showing the neurovirulence of JE14-14-2 K136E and K166Q mutants.
  • the infected mice were monitored daily for signs of morbidity and mortality. The survival curves are presented.
  • the present invention in general relates to the construction and
  • the pLAV platform may be used as a universal technology to deliver LAVs for plus-strand RNA viruses.
  • Plasmid DNA platform represents an attractive means to launch LAVs
  • DNA platform has the advantages of chemical stability, easy production, long shelf life, and no“cold chain” requirement during manufacture, transportation, and storage.
  • pLAV must enter cell membrane and nucleus membrane before it can be transcribed by eukaryotic DNA-dependent RNA polymerase; compared with the RNA platform, the extra delivery barrier of nucleus membrane may contribute to a higher dose requirement for the DNA platform.
  • the progress on DNA delivery device has enabled many DNA subunit vaccines to clinical trials. Besides
  • DNA plasmid has been reported to launch LAVs for Kunjin virus [70], YFV 17D [71], JEV SA14-14-2 [72], chikungunya virus (CHIKV) 181/clone25 strain [73], and VEEV TC-83 strain [74]
  • Kunjin virus 70
  • YFV 17D 72
  • JEV SA14-14-2 72
  • chikungunya virus 73
  • VEEV TC-83 strain VEEV TC-83 strain
  • An“adjuvant” refers to a substance that enhances an immune response, e.g., an antibody or cell-mediated immune response against a specific agent, e.g., an antigen, or an infectious agent.
  • An“attenuated” or“live-attenuated” virus strain refers to a mutated, modified, variant and/or recombinant virus having reduced or no virulence or pathogenicity or propensity to cause a disease or infection normally associated with the wildtype or unmodified, non-mutated virus.
  • an“attenuated” or“live-attenuated” virus has been modified to decrease or eliminate its pathogenicity, while maintaining its viability for replication within a target host and while remaining sufficiently
  • an “attenuated” or“live-attenuated” vaccine is a vaccine comprising an attenuated virus.
  • cDNA is DNA that is complementary to a given RNA which serves as a template for synthesis of the cDNA in a reaction that is catalyzed by reverse transcriptase.
  • cDNA is synthesized from a single stranded RNA and may be artificially produced or naturally produced by retroviruses.
  • a viral“cDNA clone”,“cDNA copy” or“DNA copy” is a double-stranded DNA copy of all or a portion of a viral genome, such as the RNA genome of an
  • a cDNA clone is generally carried in a plasmid vector.
  • A“DNA plasmid-launched live-attenuated vaccine” or“DNA-launched live- attenuated vaccine” is a DNA vaccine that comprises a cDNA copy of a live- attenuated viral genome, such as the genome of a live-attenuated plus-sense single stranded RNA virus (e.g. ZIKV, yellow fever virus or Japanese encephalitis, et al.).
  • a live-attenuated plus-sense single stranded RNA virus e.g. ZIKV, yellow fever virus or Japanese encephalitis, et al.
  • a DNA-launched live-attenuated vaccine generally includes a eukaryotic promotor, and transcribes the full-length genomic RNA of the live-attenuated virus. The full- length viral RNA then initiates replication of live-attenuated virus in the tissues of the vaccine recipient which results in efficient immunization.
  • the DNA-launched live- attenuated vaccine can also be used to prepare live-attenuated virus in vitro.
  • Heterologous means derived from a genetically distinct entity from the rest of the entity to which it is being compared.
  • a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
  • the polynucleotides of the invention may comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5'UTR, 3'UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • additional sequences such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5'UTR, 3'UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • identity refers to an exact nucleotide-to- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, introducing gaps if necessary to achieve the maximum percent identity, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. The determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.
  • Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, BLASTN, BLASTP, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods. Depending on the application, the "percent identity" can exist over a region of the sequences being compared, or, alternatively, exist over the full length of the two sequences to be compared. A skilled artisan would understand that for purposes of determining sequence identity when comparing a DNA to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide.
  • An“immunogenic composition” herein refers to a composition containing a DNA-launched live attenuated vaccine according to the invention which elicits an immune response in a susceptible host, e.g., an antibody, Th1 or cellular (e.g., T cell-mediated) immune response.
  • a susceptible host e.g., an antibody, Th1 or cellular (e.g., T cell-mediated) immune response.
  • an "isolated" biological component refers to a component that has been substantially separated or purified away from its environment or other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.
  • nucleic acid and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide may comprise modified
  • nucleotides such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
  • sequence of nucleotides may be further modified after
  • polynucleotide can be obtained by chemical synthesis or derived from a microorganism.
  • gene is used broadly to refer to any segment of polynucleotide associated with a biological function.
  • genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression.
  • gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
  • A“pharmaceutically acceptable carrier” or“excipient” refers to compounds or materials conventionally used in immunogenic or vaccine compositions during formulation and/or to permit storage.
  • A“plus-sense” or“positive sense” single stranded RNA virus is a virus that uses positive sense, single-stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA.
  • the positive-sense viral RNA genome can also serve as messenger RNA and can be translated into protein in the host cell.
  • RNA viruses account for a large fraction of known viruses, including many pathogens such as the ZIKV, chikungunya virus (CHIKV), Venezuelan equine encephalitis virus (VEEV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), hepatitis C virus, West Nile virus (WNV), dengue virus, and SARS and MERS coronaviruses, as well as less clinically serious pathogens such as the rhinoviruses that cause the common cold.
  • pathogens such as the ZIKV, chikungunya virus (CHIKV), Venezuelan equine encephalitis virus (VEEV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), hepatitis C virus, West Nile virus (WNV), dengue virus, and SARS and MERS coronaviruses, as well as less clinically serious pathogens such as the rhinoviruses that cause the common cold.
  • “Prophylactically effective amount” of a vaccine according to the invention refers to an amount sufficient to prevent or reduce the incidence of infection in a susceptible host, optionally as low as 300 or 500 ng of virus.
  • nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which does not occur in nature or by virtue of its origin or manipulation is associated with or linked to another polynucleotide in an arrangement not found in nature.
  • recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • A“susceptible host” herein refers to a host or animal that may be infected by ZIKV or other pathogenic plus-sense single stranded RNA virus(es). Such hosts include humans or animals, e.g., a human, nonhuman primate, ape, monkey, horse, cow, carabao, goat, sheep, duck, bat, or other suitable non-human host.
  • “Therapeutically effective amount” of a vaccine according to the invention refers to an amount sufficient to treat infection or disease associated therewith a plus-sense single stranded RNA virus such as ZIKV in a susceptible host.
  • A“vaccine” composition herein refers to a composition containing a DNA- launched live-attenuated vaccine according to the invention which elicits a therapeutic or prophylactic immune response against a plus-sense single-stranded RNA virus such as ZIKV, YFV or JEV.
  • variants and mutant refer to biologically active derivatives of the reference molecule that retain or enhance the desired activity, such as the ability to induce an immune response while reducing or eliminating virulence and
  • variant and mutant in reference to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that differs from the
  • sequences of such variants and mutants of the invention will have a high degree of sequence identity to the reference or corresponding wildtype sequence, e.g., a nucleic acid or amino acid sequence identity of at least 40, 50, 60, 70, 80 or 85%, more particularly at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, when the two sequences are aligned.
  • the nucleic acid sequence of the ZIKV cDNA copy that is a variant of the FSS13025 strain will generally have at least 40, 50, 60, 70, 80 or 85%, more particularly at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleic acid sequence of the wildtype FSS13025.
  • the nucleic acid sequence of the YFV cDNA copy that is a variant of the YF17D strain will generally have at least 40, 50, 60, 70, 80 or 85%, more particularly at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleic acid sequence of the wildtype YF17D strain.
  • the nucleic acid sequence of the JEV cDNA copy that is a variant of the JE14-14-2 strain will generally have at least 40, 50, 60, 70, 80 or 85%, more particularly at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleic acid sequence of the wildtype JE14-14-2 strain.
  • the "variant" or “mutant" polypeptide sequence will include the same number of amino acids as the wildtype polypeptide but will include particular substitutions, as explained herein.
  • polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless,“variant” or“mutant” polynucleotides that vary due to
  • The“variant" or “mutant” polypeptide sequence can include amino acid substitutions that are conservative in nature, i.e. , those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1) acidic-aspartate and glutamate; (2) basic-lysine, arginine, histidine; (3) non-polar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine.
  • Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity.
  • one of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.
  • ZIKV infection or“infection elicited by ZIKV” herein refers to the infection of a susceptible host with ZIKV and diseases associated therewith, including congenital ZIKV syndrome and Guillan-Barre syndrome (GBS).
  • GRS Guillan-Barre syndrome
  • YFV infection or“infection elicited by YFV” herein refers to the infection of a susceptible host with YFV and diseases and symptoms associated therewith such as fever or weight loss.
  • JEV infection or“infection elicited by JEV” herein refers to the infection of a susceptible host with JEV and diseases and symptoms associated therewith such as encephalitis, fever or weight loss.
  • the present invention provides novel DNA- launched live-attenuated vaccines for pathogenic plus-sense single stranded RNA viruses.
  • the invention relates to the construction and characterization of a vaccine comprising a DNA copy of the genome of a plus-sense single stranded RNA virus that has been modified to reduce or eliminate virulence while maintaining viability and immunogenicity (i.e. a live-attenuated virus).
  • the DNA-launched live-attenuated vaccines can be used for treating diseases related to ZIKV, YFV or JEV or providing immunoprotection against infections elicited by ZIKV, YFV or JEV, including congenital ZIKV
  • the vaccine may also prevent viremia in pregnant women and travelers to epidemic/endemic regions, avert congenital ZIKV syndrome, yellow fever or encephalitis and/or may also be useful to suppress epidemic transmission.
  • the genome of the live-attenuated plus-sense single stranded RNA virus, and the DNA copy thereof comprised in the DNA vaccine includes mutations that result in the attenuation. These mutations in particular may include mutations in the 3’UTR of the viral genome.
  • the vaccine comprises a DNA copy of a deletion variant of ZIKV, YFV or JEV wherein a portion of the 3’UTL is deleted.
  • the deletion variant of ZIKV, YFV or JEV may comprise a 20-nucleotide deletion of the 3’UTL nucleotides, e.g., at positions 10640 to 10659 of the full-length ZIKA genome. Additional suitable live-attenuated ZIKV variants comprising 3’UTL deletions are described in WO2018/152158, the entire contents of which are incorporated by reference herein.
  • the live-attenuated vaccines may be derived from different plus-sense single-stranded RNA viral strains other than those embodied herein.
  • the virus is a live-attenuated flavivirus.
  • Flaviviruses include ZIKA virus (ZIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), or tick-borne encephalitis virus (TBEV).
  • suitable viruses include any strains which are known and available in the art. Generally, the viral genomes and cDNA clones thereof will comprise the entire viral genome (modified to include the attenuating mutations).
  • the genomic sequences will have at least 40, 50, 60, 70, 80 or 85%, more particularly at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, sequence identity to a wildtype genomic sequence of the corresponding virus.
  • the DNA-launched vaccines may be derived from any ZIKV, YFV or JEV and thus may comprise DNA copies of the genomes of attenuated variants of any strain of ZIKV, YFV or JEV.
  • the source of the ZIKV DNA copy can be an attenuated variant of any one of the following strains: MR766-NIID, P6-740, ArD71 17, lbH_30656, ArB1362, ARB13565, ARB7701 , ARB15076, ArD_41519, ArD128000, ArD158084, ArD157995, FSM, FSS13025, PHL/2012/CPC-0740-Asian, H/PF/2013, PLCal_ZV, Haiti/1225/2014, SV0127_14_Asian, Natal_RGN_Asian, Brazil_ZKV2015_Asian, ZikaSPH2015, BeH815744, BeH819015, BeH819966, BeH823339, BeH828305, SSABR1 -Asian, FLR, 103344, 8375, PRVABC59, Z1 106033, MRS_OPY_Martinique,
  • VE_Ganxian_Asian, GD01_Asian, GDZ16001 , ZJ03, Rio-111 or Rio-S1 ZIKV strains (see Wang L, et al. Cell Host Microbe. 2016 May 1 1 ;19(5):561-5).
  • the strain is a North American strain or a South American strain.
  • Preferred ZIKV strains used to produce the DNA-launched live-attenuated vaccine according to the invention are FSS13025 or PRVABC59.
  • An exemplary attenuated JEV strain comprises JE14-14-2 (see Fig. 18) and an exemplary attenuated YFV strain comprises YF17D.
  • the present invention includes expression vectors that comprise the cDNA copy of a live-attenuated virus genome of the invention.
  • expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for RNA transcription and protein expression.
  • Other suitable vectors would be apparent to persons skilled in the art.
  • a cDNA copy of a live-attenuated virus genome for use in the invention in a vector is operably linked to control sequence(s) which can provide for transcription of the RNA virus and expression of the coding sequence by the vaccine recipient.
  • control sequence(s) which can provide for transcription of the RNA virus and expression of the coding sequence by the vaccine recipient.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.
  • Promoters and other expression regulation signals may be selected to be compatible with the recipient for which expression is designed.
  • mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, and the pactin promoter.
  • Viral promoters such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, rous sarcoma virus LTR promoter, adenovirus promoter), or a HPV promoter, particularly the HPV upstream regulatory region (URR) may also be used. All these promoters are readily available in the art.
  • the cDNA copy of the live-attenuated plus-sense single stranded RNA virus is contained in a plasmid, which optionally comprises a promoter, a ribosome sequence and/or a polyadenylation (pA) signal sequence.
  • the promoter may comprise a mammalian promoter.
  • the promoter can be at the 5’ end of the cDNA clone, and is optionally a simian virus 40 (SV40) or a cytomegalovirus (CMV) promoter.
  • the ribozyme sequence can be at the 3’ end of the cDNA clone, and is optionally a hepatitis delta virus ribozyme (HDVr) sequence.
  • the pA signal sequence can be at the 3’ end of the ribosome sequence, and is optionally a SV40 pA signal element.
  • the plasmid is an expression vector, optionally a mammalian expression vector.
  • the DNA-launched live-attenuated vaccines of the invention may be further modified, engineered, optimized, or appended in order to provide or select for further attenuation, immunogenicity, increased yield and/or other various features.
  • the DNA copies of live attenuated ZIKV, YFV or JEV strains or other plus-sense single stranded RNA virus strains of the invention may comprise mutations to the viral genome.
  • a mutation can be, but is not limited to, a deletion of non-coding or coding nucleotides, a deletion of one or more amino acids, an addition of one or more amino acids, a substitution (conserved or non-conserved) of one or more amino acids or a combination thereof.
  • the DNA copy of the virus can be mutated, e.g., using deletions to the 3’UTR, such that the infectivity and/or pathogenicity, of the virus is reduced.
  • the infectivity of the virus is reduced by a factor of at least 5, 10, 50, 100, 500, 10, 5x10 3 , 10 4 , 5x10 4 , 10 5 , 5x10 5 , or at least 10 6 .
  • the DNA copy of the virus genome can be mutated, e.g., having deletions to the 3’UTR and/or using point mutations, such that the rate of replication of the resulting recombinant virus is reduced or increased.
  • the rate of replication can be determined by any standard technique known to the skilled artisan.
  • the rate of replication is represented by the growth rate of the virus and can be determined by plotting the viral titer over the time post infection. The viral titer can be measured by any technique known to the skilled artisan. In certain embodiments, a
  • the suspension containing the virus is incubated with cells that are susceptible to infection by the virus including, but not limited to, Vero cells, LLC-MK-2 cells, Hep-2 cells, LF 1043 (HEL) cells, MRC-5 cells, WI-38 cells, tMK cells, 293 T cells, QT 6 cells, QT 35 cells, or chicken embryo fibroblasts (CEF).
  • the virus comprises a reporter gene.
  • the number of cells expressing the reporter gene is representative of the number of infected cells.
  • the virus comprises a heterologous nucleotide sequence encoding mCherry, and the number of cells expressing mCherry, i.e. , the number of cells infected with the virus, is determined using FACS.
  • the assays described herein may be used to assay viral titre over time to determine the growth characteristics of the virus.
  • the viral titre is determined by obtaining a sample from the infected cells or the infected subject, preparing a serial dilution of the sample and infecting a monolayer of cells that are susceptible to infection with the virus at a dilution of the virus that allows for the emergence of single plaques. The plaques can then be counted and the viral titre express as plaque forming units per milliliter of sample.
  • the viral titre express as plaque forming units per milliliter of sample.
  • the growth rate of a virus of the invention in a subject is estimated by the titer of antibodies against the virus in the subject.
  • the antibody titer in the subject reflects not only the viral titer in the subject but also the antigenicity. If the antigenicity of the virus is constant, the increase of the antibody titer in the subject can be used to determine the growth curve of the virus in the subject.
  • the growth rate of the virus in animals or humans is best tested by sampling biological fluids of a host at multiple time points post-infection and measuring viral titer.
  • the expression of heterologous gene sequence in a cell culture system or in a subject can be determined by any technique known to the skilled artisan.
  • the expression of the heterologous gene is measured by quantifying the level of the transcript.
  • the level of the transcript can be measured by Northern blot analysis or by RT-PCR using probes or primers, respectively that are specific for the transcript.
  • the transcript can be distinguished from the genome of the virus because the virus is in the antisense orientation whereas the transcript is in the sense orientation.
  • the expression of the heterologous gene is measured by quantifying the level of the protein product of the heterologous gene.
  • the level of the protein can be measured by Western blot analysis using antibodies that are specific to the protein.
  • the DNA copy of the live attenuated plus-sense single stranded RNA virus of the present invention, vectors encoding the same, and cells comprising the same may be further modified, engineered, optimized, or appended in order to provide or select for various features in addition to attenuation.
  • the attenuated virus may also contain other mutations including, but not limited to, replacing a gene of the human virus with the analogous gene of a virus of a different species, of a different subgroup, or of a different variant.
  • one or more missense mutations, additions, substitutions, or deletions can be introduced into the C, prM, E, NS1 , NS2A, NS2B, NS3, NS4A, NS4B, or NS5 proteins of the recombinant virus.
  • a deletion mutation in any of the C, prM, E, NS1 , NS2A, NS2B, NS3, NS4A, NS4B, or NS5 proteins may be introduced.
  • a missense mutation may be introduced which results in a cold-sensitive mutation or a heat-sensitive mutation.
  • major phosphorylation sites of viral protein may be removed.
  • deletions are introduced into the DNA copy of the genome of the recombinant virus.
  • a deletion can be introduced into the C, prM, E, NS1 , NS2A, NS2B, NS3, NS4A, NS4B, or NS5 proteins of the recombinant virus.
  • the intergenic region of the DNA copy of the recombinant virus is altered.
  • the length of the intergenic region is altered.
  • the intergenic regions may be shuffled from the 5' to 3' end of the viral genome.
  • the genome position of a gene or genes of the recombinant virus can be changed.
  • Attenuation of the virus is further enhanced by replacing a gene of the wild type virus with a gene of a virus of a different species, of a different subgroup, or of a different variant.
  • the attenuated phenotypes of a recombinant virus transcribed from the vaccine of the invention can be tested by any method known to the artisan.
  • a candidate virus can, for example, be tested for its ability to infect a host or for the rate of replication in a cell culture system.
  • growth curves at different temperatures are used to test the attenuated phenotype of the virus.
  • an attenuated virus can grow at 35° C., but not at 39° C. or 40° C.
  • different cell lines can be used to evaluate the attenuated phenotype of the virus.
  • an attenuated virus may only be able to grow in monkey cell lines but not the human cell lines, or the achievable virus titers in different cell lines are different for the attenuated virus.
  • viral replication in the respiratory tract of a small animal model including but not limited to, hamsters, cotton rats, mice and guinea pigs, is used to evaluate the attenuated phenotypes of the virus.
  • the immune response induced by the virus including but not limited to, the antibody titers (e.g , assayed by plaque reduction neutralization assay or ELISA) is used to evaluate the attenuated phenotypes of the virus.
  • the ability of the recombinant virus to elicit pathological symptoms in an animal model can be tested.
  • a reduced ability of the virus to elicit pathological symptoms in an animal model system is indicative of its attenuated phenotype.
  • the candidate viruses are tested in a monkey model for nasal infection, indicated by mucous production.
  • sucrose gradients and neutralization assays can be used to test the safety.
  • a sucrose gradient assay can be used to determine whether a heterologous protein is inserted in a virion. If the heterologous protein is inserted in the virion, the virion should be tested for its ability to cause symptoms even if the parental strain does not cause symptoms. Without being bound by theory, if the heterologous protein is incorporated in the virion, the virus may have acquired new, possibly pathological, properties.
  • Vaccines produced according to the invention will be used to confer prophylactic or therapeutic protection in susceptible hosts against viral infection, e.g. to treat or prevent ZIKV, YFV or JEV infection and/or to prevent congenital ZIKV syndrome or GBS or to prevent yellow fever infection or Japanese
  • the vaccines of the invention may be formulated using known techniques for formulating DNA vaccines or immunogenic compositions of DNA vaccines.
  • the vaccines of the present invention can be administered to a recipient by any available technique.
  • the vaccines may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly.
  • the vaccines may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated DNA delivery (PMDD).
  • PMDD particle-mediated DNA delivery
  • Suitable techniques for introducing DNA vaccines into a recipient include topical application with an appropriate vehicle.
  • the vaccines may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.
  • the vaccine may be present together with a pharmaceutically acceptable excipient, such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • DNA uptake may be further facilitated by addition of facilitating agents such as bupivacaine to the composition.
  • Other methods of administering DNA directly to a recipient include ultrasound, electrical stimulation, electroporation and
  • Uptake of DNA vaccines may be enhanced by several known transfection techniques, for example those including the use of transfection agents such as cationic agents, for example, calcium phosphate and DEAE-Dextran and
  • lipofectants for example, lipofectam and transfectam.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intra urethra I, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi- dose containers containing a preservative. Formulations for parenteral
  • formulations include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to,
  • a formulation for parenteral administration the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation.
  • Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • oral refers to administration of a compound or composition to an individual by a route or mode along the alimentary canal.
  • oral routes of administration of a vaccine composition include, without limitation, swallowing liquid or solid forms of a vaccine composition from the mouth,
  • a vaccine composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a vaccine composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.
  • the formulated vaccine-containing composition is suitable for intranasal, injection, topical or oral administration, for example as a dried stabilized powder for reconstitution in a suitable buffer prior to administration or in an aerosol composition.
  • the composition is injected or intranasaily administered.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, semi-solids, monophasic compositions, multiphasic compositions (e.g., oil-in-water, water-in-oil), foams microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders (see, e.g., Reference 35, Taglietti et al. (2008) Skin Ther. Lett. 13:6-8).
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carder compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such procedures are well known in the pharmaceutical industry.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • compositions may contain additional, compatible, pharmaceutically- active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • compositions of the present invention may include excipients known in the art.
  • excipients used for vaccine formulation such as adjuvants, stabilizers, preservatives, and trace products derived from vaccine manufacturing processes include but are not limited to: Aluminum Hydroxide, Amino Acids,
  • Benzethonium Chloride Formaldehyde or Formalin, Inorganic Salts and Sugars, Vitamins, Asparagine, Citric Acid, Lactose, Glycerin, Iron Ammonium Citrate, Magnesium Sulfate, Potassium Phosphate, Aluminum Phosphate, Ammonium Sulfate, Casamino Acid, Dimethyl-betacyclodextrin, 2-Phenoxyethanol, Bovine Extract, Polysorbate 80, Aluminum Potassium Sulfate, Gelatin, Sodium Phosphate, Thimerosal, Sucrose, Bovine Protein, Lactalbumin Hydrolysate, Formaldehyde or Formalin, Monkey Kidney Tissue, Neomycin, Polymyxin B, Yeast Protein, Aluminum Hydroxyphosphate Sulfate, Dextrose, Mineral Salts, Sodium Borate, Soy Peptone, MRC-5 Cellular Protein, Neomycin Sulfate, Phosphate Buffers, Polysorbate,
  • the vaccine or immunogenic composition may be used in the vaccination of a mammalian recipient or host, particularly a human, nonhuman primate, ape, monkey, horse, cow, carabao, goat, duck, bat, or other suitable non-human host.
  • a mammalian recipient or host particularly a human, nonhuman primate, ape, monkey, horse, cow, carabao, goat, duck, bat, or other suitable non-human host.
  • the subject may be immunocompromised or may have another condition, e.g., may be pregnant.
  • Vaccines are the most effective means to fight and eradicate infectious diseases.
  • Live-attenuated vaccines usually have the advantages of single dose, rapid onset of immunity, and durable protection.
  • DNA vaccines have the advantages of chemical stability, ease of production, and no cold chain requirement. The ability to combine the strengths of LAV and DNA vaccines may transform future vaccine development by eliminating cold chain and cell culture with the potential for adventitious agents.
  • a DNA-launched LAV was developed for Zika virus (ZIKV), a pathogen that recently caused a global public health emergency.
  • ZIKV Zika virus
  • a cDNA copy of a ZIKV LAV genome was engineered into a DNA plasmid.
  • the DNA- LAV plasmid was delivered into mice using a clinically proven device TriGridTM to launch the replication of LAV.
  • a DNA- launched live-attenuated vaccine (LAV) that combines the advantages of DNA vaccines (chemical stability, no cold chain, easy production, and low cost) and LAVs (single dose, quick immunity and durable protection).
  • LAV DNA- launched live-attenuated vaccine
  • a single-dose vaccination as low as 0.5 pg of the DNA-LAV plasmid elicited 100% protective immunity within 14-21 days in A129 mice.
  • the vaccination completely prevented ZIKV infection, in utero transmission during pregnancy, and male reproductive tract infections.
  • the immunized mice also developed robust T cell responses.
  • DNA-LAV approach may serve as a universal vaccine platform for other plus-sense RNA viruses, e.g., other flaviviruses. This is highly significant as enhancing vaccine performance with improved simplicity and immunity is critical, particularly when responding to epidemic emergencies. The ability to combine the advantages of different vaccine platforms could transform future vaccine development.
  • the African green monkey kidney epithelial (Vero) cell and human embryonic kidney cell (293T) were purchased from the American Type Culture Collection (ATCC, Bethesda, MD) and maintained in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, South Logan, UT) and 1%
  • P/S penicillin/streptomycin
  • mAb mouse monoclonal antibody
  • ATCC flavivirus E protein
  • ZIKV NS5 mouse polyclone antibody against ZIKV NS5 (in-house generated using the recombinant ZIKV NS5 protein purified from E.coli.)
  • ZIKV-specific HMAF hyper-immune ascitic fluid; obtained from the World Reference Center of Emerging Viruses and
  • Plasmid Construction The plasmid pFLZIKV-PRV (derived from a singlecopy vector pCC1TM [Epicentre, Madison, Wl]) [28] was used as a starting vector to construct the DNA-launched plasmids in this study. Firstly, the cDNA sequence of ZIKV strain Cambodian FSS13025 (GenBank accession No.
  • KU955593 KU955593
  • HDVr hepatitis delta virus ribozyme
  • SV40 simian virus 40
  • CMV cytomegalovirus
  • the resulting DNA fragments were cloned into the pCC1-T7- ZIKV plasmid using restriction enzymes Hpal and Nhel, resulting in subclones pCC1-SV40-ZIKVa and pCC1-CMV-ZIKVa.
  • SV40 or bovine growth hormone (BGH) polyadenylation (pA) signal sequences were amplified from the pcDNA3.1 vector and cloned into the pCC1-SV40-ZIKVa and pCC1-CMV-ZIKVa through restriction enzymes Clal and Srfl, respectively, resulting in plasmids pSV40- ZIKV (short as WT or SV40-WT) and pCMV-ZIKV (short as CMV-WT).
  • BGH bovine growth hormone
  • pA bovine growth hormone
  • the flavivirus-conserved polymerase motif GDD mutation (corresponding to residues Gly664, Asp665, and Asp666 in ZIKV NS5 were mutated to Ala) [30] and the 3’UTR 20 nucleotide deletion (D20) [22] was introduced by overlap PCR and cloned into the plasmid pCC1-SV40-ZIKV through restriction enzymes EcoRI and Clal, resulting in plasmids pFLZIKV-AGDD (short as AGDD) and pFLZIKV-3'UTR-A20 (short as D20). Plasmids were propagated in the TransforMax EPI300 Chemically Competent E.
  • This pCC1TM vector-derived plasmid could be induced to generate 10-20 copies/cell using L-arabinose in the E. coli strain EPI300. All restriction enzymes were purchased from New England BioLabs (Ipswitch, MA). All plasmids were validated through restriction enzyme digestion and Sanger DNA sequencing. All primers were synthesized from Integrated DNA Technologies (Skokie, Illinois) and available upon request.
  • Viral titers were determined by plaque assay.
  • Plaque assay 1.5x10 5 Vero cells per well were seeded into a 24-well plate. The next day, 100 pi of undiluted virus sample or series of 10-fold diluted virus samples were added to individual well of cell monolayer. After 1 h of incubation at 37°C with 5% CO2, the inoculum in each well was replaced with 0.6 ml of overlay medium (DMEM medium supplemented with 2% FBS and 0.8% methylcellulose [Sigma]). After incubation at 37°C with 5% CO2 for 4 days, cells were fixed in 3.7% formalin solution and stained with 1% crystal violet. For ZIKV D20 mutant viruses, viral titers were determined by focus-forming assay as described previously [22]
  • IFA Immunofluorescence assay
  • 8x10 4 Vero Cells were seeded into each well of an 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific). The next day, cells were transfected with 0.5 pg of DNA per well. At selected time points, cells were fixed with chilled methanol at -20°C for 30 min. After 1 h incubation in blocking buffer (PBS supplemented with 1 % FBS and 0.05% Tween-20), cells were incubated with the primary antibody 4G2 for 1 h. After three PBS washes, cells were incubated with goat anti-mouse IgG conjugated with Alexa Fluor 568 (1 :1000 diluted in blocking buffer) for 1 h. Finally, after three PBS washes, cells were mounted in a Vectashield mounting medium with DAPI (Vector Laboratories). Fluorescence images were acquired under Eclipse Ti2 inverted fluorescence microscope (Nikon Instruments Inc.).
  • Lysates were centrifuged at 20,000 x g and 4°C for 30 min to remove cell debris. Supernatants were collected and mixed with 4 c LDS sample buffer (ThermoFisher Scientific). After denaturing at 70°C for 15 min, 10 pi samples were loaded onto to a 12% Mini-Protean TGX Stain-Free Precast gel (Bio-Rad Laboratories). After separation by electrophoresis, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories).
  • PVDF polyvinylidene difluoride
  • the blot was firstly incubated at room temperature for 1 h in a blocking buffer containing TBST (10 mM Tris-HCI [pH 8.0], 150 mM NaCI, and 0.1% Tween 20) and 5% skim milk, followed by 1 h of incubation with primary antibody
  • RT-PCR and Sequencing Viral RNAs in culture fluids (140 mI) or mouse serum were used for viral RNA extraction by QIAamp viral RNA mini kit (Qiagen). RT-PCR assays were performed using Superscript III One-Step RT-PCR System with Platinum Taq DNA Polymerase kit (Life technologies) following the
  • Plasmid DNA was diluted to indicated concentration in calcium/magnesium-free phosphate- buffered saline (DPBS, ThermoFisher Scientific) and administrated into A129 mice by intramuscular (IM) injection or by IM injection together with electroporation (IM&EP) using the TriGridTM Delivery System (Ichor Medical Systems, San Diego, CA) as described previously [31].
  • DPBS calcium/magnesium-free phosphate- buffered saline
  • IM&EP electroporation
  • mice are a model susceptible to ZIKV infection [32]
  • TriGridTM device For consistent dosing by TriGridTM device, six-week-old mice A129 mice with weight above 15 g were chosen for this study. Briefly, after anesthetized with isoflurane gas, mice were injected into one tibialis anterior muscle with 20 pi of DNA solution using a 3/10 ml U-100 insulin syringe (Becton-Dickinson, Franklin Lakes, NJ) inserted into the center of a TriGrid electrode array with 2.5 mm electrode spacing. Mock-infected mice were given DPBS by the same route. Injection of DNA was followed immediately by electrical stimulation at an amplitude of 250 V/cm, and the total duration was 40 ms over a 400-ms interval. The control intramuscular injection was performed as described above without the application of electrical stimulation.
  • mice were monitored for weight loss and signs of disease daily. At selected time points, mice were bled via the retro-orbital sinus (RO) and viremia was determined by plaque assay. Neutralizing antibodies in sera were measured using ZIKV/mCherry infection assay. Mice were challenged on day 29 post-immunization with parental ZIKV strain PRVABC59 (10 6 PFU) via the subcutaneous route. On day 2 post challenge, mice were bled and viremia was determined by plaque assay. Sperm counting was performed according to the protocol as described previously [23] Mice were euthanized and necropsied at indicated time points. Epididymis and testes were harvested immediately. Motile and non-motile sperms were counted manually on a hemocytometer by microscopy. Total sperm counts equal to the sum of motile and non-motile sperms.
  • RO retro-orbital sinus
  • mice were bled for measuring NTso. Mice were mated starting on day 30 post-immunization.
  • Mouse embryonic development started (E0.5) once mouse vaginal plugs were observed.
  • mice were challenged with parental ZIKV strain PRVABC59 (10 6 PFU) via the subcutaneous route.
  • mice were bled to measure viremia.
  • E18.5 all dams were euthanized and maternal tissues (brain, spleen and placenta) and fetuses were harvested. Fetal weight was measured immediately.
  • fetal heads and blood were collected.
  • Mouse tissues were homogenized in 500 pi of DMEM medium using TissueLyser II (Qiagen) for 5 min at 30 Flz. After centrifugation at 15,000*rpm for 10 min, supernatants were harvested. Plaque assays were performed on Vero cells to determine virus loads in maternal brain, spleen and placenta, and fetal head.
  • Neutralizing antibodies in fetal serum were measured using ZIKV/mCherry neutralization assay as described above.
  • ICS Intracellular cytokine staining
  • splenocytes were stimulated with 1 x10 5 IFU of live ZIKV (strain FSS13025) for 24 h or 10 pg/ml E peptide (Sequence 294-302 in ZIKV polyprotein) [33] for 5 h.
  • Live ZIKV was used as a stimulant for measuring both CD4 + and CD8 + T cell response [34]
  • the E peptide was used as stimulant for measuring CD8 + T cell response [33].
  • BD GolgiPlug (BD Bioscience) was added to block protein transport.
  • Cells were stained with antibodies against surface markers CD3 (APC- conjugated) and CD4 (FITC-conjugated) or CD8 (FITC-conjugated). Afterwards, cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin. Cells were then incubated with PE-conjugated anti-IFN-g and PE-Cy7-conjugated anti-TNF-a antibodies or control PE-conjugated rat lgG1. Samples were processed with a BD AccuriTM C6 Flow Cytometer instrument. Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).
  • Bio-Plex immunoassay Approximately 3x10 5 splenocytes per well were plated in a 96-well plate and stimulated with 2 c 10 4 FFU of ZIKV (strain FSS13025) for 2 days, respectively. Culture supernatants were harvested and frozen at -80°C. Cytokines IL-2, IFN-Y and TNF-a in the culture supernatants were measured using a Bio-Plex Pro Mouse Cytokine Assay (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.
  • ZIKV-3’UTR-A20 (a LAV candidate containing a 20-nucleotide deletion within the 3’UTR of the ZIKV genome) into a plasmid DNA-launched LAV.
  • ZIKV-3’UTR-A20 has an excellent safety and efficacy profile: a single-dose vaccination of 10 3 FFU confers sterilizing immunity in NHPs [23]
  • To convert it to a plasmid-launched LAV we selected the pCC1TM vector to clone the cDNA of ZIKV- 3'UTR-A20 because its copy number can be conditionally controlled in E. coir (i) A single copy per cell to maximize the plasmid stability during cloning and (ii) 10-20 copies per cell to maximize plasmid yield during production [35]
  • a eukaryotic promoter was engineered at the 5' end of ZIKV-3'UTR-A20 cDNA to launch the transcription of viral RNA through cellular RNA polymerase II (Fig. 1A).
  • a hepatitis delta virus ribozyme (HDVr) sequence and a polyA-signal sequence were engineered at the 3' end of ZIKV-3'UTR-A20 cDNA for generation of the authentic 3' end of the viral RNA and for transcription termination (Fig. 1A).
  • the resulting plasmid is named as pZIKV-3'UTR-A20.
  • pZIKV-3'UTR-A20 Once the SV40-driven pZIKV-3'UTR-A20 was constructed, we characterized its ability to launch replication of the LAV virus in cell culture. Upon transfection into Vero cells, pZIKV-3'UTR-A20 generated viral E protein-positive cells (FIG. 1B), viral NS5 protein (FIG. 1C), and high titers of LAV virus (peak viral titer of 2x10 6 PFU/ml; FIG. 1D). Compared with pZIKV-WT, pZIKV- 3'UTR-A20 produced fewer E-positive cells (FIG. 1B) and less NS5 protein (FIG.
  • mice were administered replication-defective pZIKV-AGDD (10 pg), which was expected to yield translation of transcribed ZIKV RNA but not subsequent viral replication, or DPBS. After immunization, all mice remained healthy with no detectable pathologic changes at the site of injection or adverse effects including no weight loss (FIG. 2B). Viremia of ⁇ 10 4 PFU/ml was detected in each of the three pZIKV-3'UTR-A20-dosed groups (FIG. 2C-F), although the 1 -pg dosed group exhibited a higher viremia-positive rate (88%) than the 0.1 -pg (25%) and 0.01-pg (27%) groups (FIG. 1 K).
  • TriGridTM for efficient DNA delivery.
  • A129 mice were intramuscularly needle injected with 1 pg of pZIKV-WT and pZIKV-3’UTR-A20, then analyzed for viremia and neutralizing antibodies (FIG. 10A).
  • FIG. 3B On day 29 post-immunization, pZIKV-3’UTR-A20 and pZIKV-WT elicited comparable levels of neutralizing antibody titers (FIG. 3B). No infectious virus was detected in brains (FIG. 3C), spleens (FIG. 3D), or testes (FIG. 3E) of pZIKV-3'UTR-A20-, pZIKV-WT- or DPBS-immunized mice. Notably, one testis from each of the pZIKV-WT-immunized animals suffered significant weight loss compared with the other testis (FIG. 3F&G).
  • mice immunized with pZIKV-3'UTR-A20 or DPBS did not exhibit any weight loss or oligospermia (FIG. 3F-I).
  • pZIKV-3'UTR-A20 immunization does not cause persistent infection or oligospermia.
  • FIG. 4A Six-week-old A129 male mice were immunized with 1 pg of pZIKV-3'UTR-A20 or DPBS. By day 29 post-immunization, pZIKV-3'UTR-A20 elicited robust neutralizing antibody titers of 10 4 (FIG. 4B). On the same day, mice were challenged with 10 6 PFU of ZIKV PRVABC59 by the subcutaneous route.
  • mice developed high neutralizing antibody titers of 5.6x10 3 on day 29 post-immunization (FIG. 5B).
  • Female mice were then mated with males on days 30- 37 post-immunization, and examined for pregnancy [indicated by vaginal plugs observed after mating and defining embryotic day 0.5 (E0.5)].
  • E0.5 embryotic day 0.5
  • the pregnant mice were challenged with 10 6 PFU of ZIKV PRVABC59 by the
  • T cell response after pZIKV-3’UTR-A20 immunization T cell immunity plays an important role in preventing ZIKV infection
  • Splenocytes were cultured ex vivo, stimulated with a previously reported ZIKV E peptide [33] or infectious WT ZIKV, and analyzed by an intracellular cytokine staining (ICS) assay and a Bio-Plex immunoassay.
  • ICS cytokine staining
  • Bio-Plex immunoassay The pZIKV-3'UTR-A20-immunized animals had significantly more ZIKV-specific IFN-y + and IFN-y + TNF-a + CD4 + (FIG 6A and FIG.
  • FIG. 11 A and CD8 + T cells (FIG. 6B and FIG. 11B&C) than the DPBS-vaccinated animals.
  • splenocytes from the pZIKV-3'UTR-A20-immunized mice produced significantly higher levels of IL-2 (Fig. 6C), IFN-g (FIG. 6D), and TNF-a (FIG. 6E) proteins than the DPBS-immunized animals upon ex vivo re-stimulation with ZIKV.
  • Vaccines, especially LAV have been highly effective in controlling and even eradicating infectious diseases
  • Enhancing vaccine performance with improved simplicity, immunity, and delivery speed is critical, particularly when responding to epidemic emergency.
  • the goal of this study is to develop and characterize pZIKV- 3'UTR-A20 that combines the strengths of DNA vaccines (chemical stability, no cold chain, easy production, and low cost) and LAVs (single dose, quick immunity and durable protection).
  • Our results showed that a single-dose vaccination of >0.5 pg of pZIKV-3'UTR-A20 elicited 100% protective immunity within 14-21 days in the A129 mice. The vaccination completely prevented ZIKV infection, vertical transmission during pregnancy, and male reproductive tract infections.
  • RT-PCR test is much more sensitive than the plaque and focus-forming assays (FIG. 12)
  • future non-human primate studies should employ RT-PCR assay to detect viral RNA.
  • the RT-PCR assay should also be used in preclinical safety studies to measure viral RNA levels in different organs collected at multiple time points post vaccination. Besides antibody response, the immunized mice also developed robust T cell responses.
  • the DNA-launched approach could serve as a universal platform to deliver LAVs for other positive-sense, single-stranded RNA viruses.
  • DNA and mRNA subunit vaccines (expressing viral prM-E proteins) have been well developed for ZIKV [14-18], among which DNA subunit vaccines have already shown promising safety and immunogenicity in phase I clinical trials [20,
  • RNA/DNA delivery methods different promoters used in plasmids and mouse strains.
  • lowering the minimal protective dose is desirable for a vaccine, particularly when responding to epidemic emergencies that often require the rapid production of millions of doses for vaccinating large populations.
  • lower doses of DNA plasmid could minimize potential adverse effects in vaccinees.
  • future studies should be performed to further improve the delivery efficiency of pZIKV-3'UTR-A20 by comparing different DNA delivery devices (e.g., injection/electroporation device from Inovio and needle-free injection device from ParmaJet) and through different routes of administration (e.g., intradermal versus intramuscular).
  • nanoparticle technology could also be explored for the efficient delivery of the DNA-launched LAVs.
  • Such nanoparticle formulations have to be co-developed with the DNA-launched LAVs in pre-clinics and clinics.
  • Engineering pZIKV-3'UTR-A20 with a reporter gene may facilitate tracking the initial production and spread of the DNA-launched LAV.
  • a reporter gene e.g., GFP or mCherry inframe fused with the viral open-reading-frame
  • the same experiment may also be used to estimate the duration of LAV production after plasmid vaccination.
  • 19A-B outlines the cloning scheme for pYF17D and pJE14-14-2.
  • the viral RNA was transcribed from a 5’ eukaryotic SV40 promoter through cellular RNA polymerase II.
  • SV40 promoter rather than CMV promoter, because SV40 was reported to more efficiently launch ZIKV pLAV (ZIKV-3'UTR-A20)
  • HDVr hepatitis delta virus ribozyme
  • polyadenylation signal were engineered at the 3' end of YF17D or JE14-14-2 cDNA to produce the authentic 3' end of viral RNA and to terminate transcription, respectively.
  • plasmid pCCITM as the vector because flavivirus cDNA is often unstable in medium and high copy number plasmids [58] Single copy vector is often essential to maintain the stability of flavivirus cDNA clones.
  • the copy number of pCCITM plasmid can be conditionally controlled: single copy per E. coli during cloning and 10-20 copies per cell during production [79]
  • FIG. 14 A129 mice (FIG. 14).
  • mice Nine-week-old mice were subcutaneously infected with 10 4 PFU of YF17D or 105 PFU of JE14-14-2. Mice inoculated with PBS were included as a control.
  • YF17D infection did not cause disease (data not shown), weight loss (FIG. 14A), or death (FIG 14B), but developed viremia that peaked on day 2 p.i. (FIG. 14C).
  • JE14-14-2 infection caused diseases (including ruffled fur, hunched posture, and squinty eyes), weight loss (FIG. 14D), viremia that peaked on day 3 p.i. (FIG. 14F), and 20% death (FIG. 14E).
  • mice were also subcutaneously immunized with 10 5 PFU of YF17D LAV.
  • mice None of the pYF17D-immunized mice developed disease, weight loss (FIG. 15B), or viremia (data not shown).
  • 100% of the mice vaccinated with 1 or 0.3 pg of pYF17D developed neutralizing antibodies with titers of ⁇ 1/1 ,000, whereas 60% (3/5) of the animals from the 0.1 pg group developed neutralizing antibodies (FIG. 15C, left panel).
  • the mice inoculated with 10 5 PFU of YF17D LAV developed neutralizing tiers of ⁇ 1/2,000.
  • the mice were bled to measure neutralizing antibody titers and subcutaneously challenged with 10 5 PFU of YF17D LAV.
  • mice developed disease or weight loss, indicating the safety of the vaccine. Mice immunized with 0.3 pg of pYF17D conferred 100% seroconversion and protection against challenge. All seroconverted animals seemed to have developed sterilizing immunity.
  • plasmids from passage 0 (P0) and 5 (P5) produced equivalent levels of LAVs after transfection with equal amounts of pLAVs (4 pg) into cells (FIG. 17B, C).
  • the LAVs produced from the P0 and P5 plasmids developed identical plaque morphologies (Fig. 5d).
  • Another distinct feature of our pLAVs is that a single-dose immunization of 300 ng DNA elicited neutralizing antibody titers that were not boosted by virus challenge, suggesting that the vaccinated mice developed a sterilizing neutralizing antibody response.
  • the antiviral immunity is determined by the kinetics of LAV production from the pLAV DNA and the immune response to the LAVs. The immune stimulation and development upon pLAV vaccination could be very different from those upon conventional LAV immunization, such differences may account for the development of sterilizing neutralizing antibody response. Further studies are needed to track the spread of the pDNA-launched LAV to antigen-presenting cells, such as dendritic cells, which are essential for the induction of antiviral response [83].
  • a pLAV with a GFP reporter may facilitate such tracking experiments.
  • the pLAV platform also minimizes the stability concern of conventional LAVs during production on cells or eggs.
  • the pYF17D and pJE14-14-2 were stable after 10 rounds of continuously culturing in E. coli (FIG. 17).
  • the DNA- launched chikungunya vaccine was reported to generate fewer reversions of the attenuating mutations compared to conventional virus administration [84]
  • cell types are important for maintaining the stability of LAVs during amplification. JE14-14-2 was much less stable when cultured on Vero cells than BHK-21 cells.
  • the most optimal delivery device will be chosen for each pLAV after comparing the performances of different devices (e.g., injection/electroporation devices such as are available from Ichor and Inovio and needle-free injection devices available from ParmaJet) and through different administration routes (e.g., intradermal versus intramuscular). Third, it is important to determine the durability of the pLAV-mediated protection.
  • injection/electroporation devices such as are available from Ichor and Inovio and needle-free injection devices available from ParmaJet
  • administration routes e.g., intradermal versus intramuscular.
  • Tretyakova, I., et al., Novel vaccine against Venezuelan equine encephalitis combines advantages of DNA immunization and a live attenuated vaccine.
  • Tretyakova, I., et al., Novel vaccine against Venezuelan equine encephalitis combines advantages of DNA immunization and a live attenuated vaccine.

Abstract

L'invention concerne d'une manière générale le développement de compositions immunogènes comprenant une copie d'ADN d'au moins un génome de virus à ARN simple brin sens positif atténué vivant, tel que le génome d'un virus Zika (ZIKV) atténué vivant, du virus de l'encéphalite japonaise (JEV) ou du virus de la fièvre jaune (YFV). Les compositions immunogènes peuvent être utilisées pour traiter ou conférer une immunité protectrice contre des maladies associées à des virus à ARN simple brin sens positif, par exemple pour permettre une immunoprotection prolongée contre de tels virus et une immunité protectrice étant conférée à une dose aussi faible que, par exemple, 300 ou 500 ng après administration d'une dose unique de pLAV.
EP19857888.2A 2018-09-04 2019-08-30 Vaccins atténués vivants à base de plasmide d'adn pour virus à arn simple brin sens positif Pending EP3846848A4 (fr)

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